Our previous approach of using mass spectral identification of co-purified proteins to identify factors that bind to STAMP was thwarted by the very low levels of endogenous STAMP and non-specific binding to transiently transfected Flag/STAMP. For this reason, we have concentrated on using microarray data from cells with and without STAMP to identify possible binding proteins. As described elsewhere (DK047039-05), we have recently prepared mouse embryo fibroblasts (MEFs) from wild type mice and mice in which the endogenous STAMP gene has been knocked out (KO mice). Microarray analyses have been performed on cells that were induced by glucocorticoid steroid for 8 hr. High quality data were obtained for almost 3,000 genes, for which the level of expression changed by ≥1.5 fold after Dex treatment. Those genes with the largest change in expression between wild type and KO MEF cells are being examined for the involvement of other known cofactors. Our hypothesis is that STAMP interaction with these known cofactors may be responsible for the significant change in glucocorticoid-regulated gene expression in the STAMP KO MEF cells. Those genes for which there are major differences in basal level expression are candidates for mediating the apparent steroid-independent effects of STAMP, such as cell growth and tumor formation (He et al., 2010, BMC Cancer, 10, 128). Initially identified cofactors will be further examined for STAMP binding by co-immunoprecipitation and for cofactor activity in gene induction assays using transiently transfected cells. At the same time, STAMP KO mice in a homogeneous genetic background (C57/BL6) have been prepared and are being studied for phenotypic abnormalities. The identification and characterization of any physiological defects will point to other proteins that might bind STAMP. Finally, studies of the transcriptional properties of the combination of GR, TIF2, and STAMP (described in DK047039-05) indicate that GR, TIF2, and STAMP can form a ternary complex in which different protein surfaces unequally modify the Amax, EC50, and PAA of steroid-regulated gene induction. These results suggest that the effects of STAMP-associated proteins can be more subtle, and selective, than initially believed. The ability to modulate one, two, or all three parameters of GR-controled gene expression heightens the importance of identifying other proteins that can bind to STAMP. These studies should identify new proteins that not only participate in, or modify the activity of, STAMP modulation of the Amax, EC50 and PAA in GR-regulated gene expression but also receptor-independent processes such as cell growth. These results will increase our understanding of several physiologically relevant transcriptional properties of GR-steroid complexes that permit a continuum of responses and constitute new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and endocrine therapies. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.
